Project Summary Desmoplastic Small Round Cell Tumor (DSRCT) is defined by a recurrent chromosomal translocation that creates a chimeric transcription factor, EWS-WT1, which is the only known oncogenic driver of DSRCT. Unfortunately, oncogenic transcription factors such as EWS-WT1 are difficult to target for therapy. Given that standard treatments are not very effective for DSRCT (less than15% 5 year survival), development of rationale-based targeted therapy is urgently needed. To do so, new vulnerable oncogenic targets downstream of EWS-WT1 must be identified. Unfortunately, not much is known about the potential vulnerable targets in DSRCT. To identify the vulnerabilities in DSRCT, we performed integrative gene expression analysis using expression profiles of EWS-WT1-depleted DSRCT cells and 28 primary DSRCT samples. This analysis identified several kinases that could be critical for DSRCT cell growth and survival. Most cancers contain a small population of tumor initiating cells (TICs) that are thought to serve as seeds for recurrent tumor growth, but TICs have not yet been identified in DSRCT. We have recently identified a novel culture condition that produces spheroids in DSRCT which display significantly higher tumor-seeding activity than the adherent cells. Deep RNA sequencing analysis of spheroids has revealed a number of kinases that are enriched in spheroids. Thus, the goal of this proposal is to identify key vulnerabilities in DSRCT that will ultimately lead to development of rationale-based targeted therapy. Aim 1. Investigate the identified kinases as potential vulnerabilities in DSRCT. We have identified NTRK3, ROCK2 and SIK1 kinases that are highly expressed in primary DSRCT tumors and in DSRCT cells. We will investigate the effects of inhibiting these kinases on DSRCT cell growth in vitro and in vivo. Aim 2. Investigate the roles of BLK, NTRK3 and ROCK2 in spheroids. Our deep RNA sequencing analysis of highly tumorigenic spheroids has revealed a number of kinases whose expression is greatly increased in spheroids. We will investigate the effects of inhibiting these kinases in tumorigenic capacity of spheroids using our orthotopic intraperitoneal DSRCT xenograft model. DSRCT remains untreatable with unacceptably high mortality. Our integrative gene expression analysis and our recent discovery of spheroids in DSRCT have revealed several kinases that could be critical for DSRCT growth or survival. These complementary approaches will ultimately lead to development of durable targeted therapies in DSRCT. Our integrative approach in identifying vulnerable targets could be applied to finding critical dependencies in other related pediatric sarcomas such as Ewing sarcoma.